1. Field of the Invention
The present invention relates to biotechnology, and specifically to a method for producing branched-chain L-amino acids. More specifically, the present invention discloses the use of a new L-valine-resistant enzyme which is involved in the biosynthesis of branched-chain L-amino acids. More specifically, the present invention concerns a L-valine-resistant mutant acetolactate synthase (AHAS I) purified from E. coli, a bacteria from the Enterobacteriaceae family which contains this synthase enzyme, and a method for producing branched-chain L-amino acids by fermentation using the strains of this bacteria.
2. Brief Description of the Related Art
Traditionally, L-amino acids have been industrially produced by fermentation utilizing strains of microorganisms obtained from natural sources, or mutants thereof which have been specifically modified to enhance L-amino acid productivity.
Many techniques have been previously described to specifically enhance L-amino acid productivity, such as, for example, transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Such techniques are based on increasing the activities of the enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to feedback inhibition by the produced L-amino acid (see, for example, Japanese Laid-open application No. 56-18596 (1981), WO 95/16042 or U.S. Pat. Nos. 5,661,012 and 6,040,160).
The biosynthesis of isoleucine, leucine, and valine occurs through a branched pathway in which three steps are common to each end product. The AHAS reaction represents the first biosynthetic step common to the three products. The reaction is catalyzed by isoenzymes which are the target of end-product inhibition by valine. This regulation plays a major role in the physiological control of the pathway in bacteria. The reaction includes condensation of active acetaldehyde (derived from pyruvate) with either α-ketobutyrate or pyruvate to yield α-aceto-α-hydroxybutyrate (a precursor of isoleucine) or α-acetolactate (a precursor of leucine and valine), respectively.
It has been reported that valine and its keto-acid precursor α-ketoisovaleric acid inhibit the growth of E. coli K12, and that isoleucine counters this inhibition (Tatum, E. L., Fed. Proc. 8:511 (1946)). At present, it is commonly accepted that inhibition of valine primarily results from blocking α-aceto-α-hydroxybutyrate synthesis. An analysis of E. coli K12 has revealed that this strain contains the structural genes for the three AHAS activities, which are designated as isoenzymes AHAS I, AHAS II, and AHAS III. AHAS I and AHAS III are both inhibited by valine, whereas AHAS II is resistant to it; however, AHAS II is not normally expressed in E. coli K12 cells (Guardiola, J. et al, Mol. Gen. Genet. 156:17-25 (1977)). All AHAS isozymes from enterobacteria are composed of a large and a small subunit in an α2β2 structure, with the large subunits performing a catalytic function and the small subunits performing a regulatory function. The small subunits are absolutely required for sensitivity of the enzyme activity to the feedback inhibitor valine. A study of the individual properties of the AHAS I and AHAS III subunits (Weinstock O. et al, J. Bacteriol. 174:5560-5566 (1992)) showed that the small subunits specifically induced a catalytically competent conformation of the whole enzyme and stabilized the transition state.
On the basis of a model of the valine-binding region of the AHAS III regulatory small subunit from E. coli, truncations from the carboxyl end of the small subunit were made. These truncations induce a lack of valine sensitivity in the truncated AHAS III enzymes (Mendel S. et al, J Mol Biol. 10; 325(2):275-84 (2003)).
But at present there are no reports describing mutant bacterial acetolactate synthase (AHAS I) which is feedback resistant to valine and the use of such a mutant acetolactate synthase for improving branched-chain L-amino acid production in corresponding L-amino acid producing strains.